Genomic equivalence

1
Lecture 8

• Genomic equivalence
• cloning

2
Examples of differentiation

• Muscles
• Blood Lymph (hematopoietic system)
• Neural crest

3
Muscle differentiation

• Focus on skeletal muscles
• all derived from somites--myotome
• somitic mesoderm generates myoblasts
• myoblasts divide until growth factors removed,
  then differentiate into muscle cells

4
President of the Regents of the University of
California
5
Muscle differentiation stages

• W Fig 9.12

6
Muscle differentiation genes

• W Fig 9.13

7
The discovery of MyoD

• Hal Weintraub 1987
• Fibroblasts in culture can be converted into
  myoblasts by 5-azacytidine treatment (causes
  demethylation)
• Infer from rate of conversion that maybe only 1
  or 2 genes need to be turned on
• Made cDNA libraries from cells before and after
  treatment
• Subtractive hybridization to find the difference
  betweenthe 2 libraries
• A few genes different
• Transfection of a single gene, MyoD, SUFFICIENT
  to convert fibroblasts to myoblasts!
• basic helix loop helix (bHLH) transcriptional
  regulator A master controller of muscle
  differentiation?

8
MyoD is sufficient but not necessary for muscles
to form

• MyoD knockout mice look normal!?
• MyoD is member of multigene family
• redundancy with Myf5
• MyoD Myf5 double knockouts lack all myoblasts

9
Rest of lecture

• Instability of the differentiated state
• A digression on exceptions to genomic constancy
• Regeneration, cloning etc

10
Instability of differentiated state-1

• Trans-differentiation (metaplasia)
• Direct conversion of 1 differentiated cell type
  to another (usually related)
• Pancreas cells convert to liver in copper
  deficiency
• Liver to pancreas conversion after PCB treatment
• Pancreatic cells marked with GFP turn on liver
  marker (red) after dexamethasone treatment

Fig 9.33
Nature Reviews Molecular Cell Biology 3, 187
-194 (2002) HOW CELLS CHANGE THEIR PHENOTYPE
David Tosh Jonathan M. W. Slack
11
Instability of differentiated state-2

• de-differentiation
• Conversion of differentiated cell type to
  pluripotent state
• In regenerating limbs, muscle dedifferentiates to
  form blastema, can make cartilage (section 13.1)
• Totipotency of differentiated cells cloning

Newt limb regeneration
Movie of regeneration http//darwin.bio.uci.edu/
mrjc/Movie/movie.html
12
genomic constancy

• are all cells in an organism genetically
  identical? yes.
• first, two exceptions
• evidence that the general rule is yes
• descriptive evidence
• functional evidence

13
chromatin diminution

• Theodor Boveri 1887 studied Parascaris univalens
  horse parasitic nematode

After 60 euchromatic (E) minichromosomes will
segregate to somatic daughter Heterochromatic
ends (H) are eliminated
Before diminution 1 pair of chromosomes (DAPI
stain for DNA)
14
chromatin diminution

• Only germline retains complete genome, somatic
  cells cannot be totipotent (?)
• special kind of cytoplasm (germ plasm) in
  germline protects DNA from degradation?
• Diminution found in some nematodes (not C.
  elegans), insects, crustaceans.In some insects
  entire chromosomes are eliminated (chromatin
  elimination)
• But these appear to be special cases

15
Second example of genomic change in
differentiation

• Antibody (Immunoglobulin) genes in vertebrates
• How are trillions of antibody types generated?
• estimated 1012 possible types of antibody, but
  we have only 105 genes?!

16
DNA rearrangement in adaptive immunity
Susumu Tonegawa (1987 Nobel Prize)
Fig 9.27

• B lymphocytes (antibody secreting cells)
• DNA undergoes irreversible rearrangement during
  maturation
• Special recombinase enzyme RAG1
• mature B-cell has less DNA than germline cell
• Also somatic hypermutation

17
do other tissues undergo DNA changes?

• T lymphocytes (T cell receptor gene
  rearrangement) TCR segments undergo
  recombination (RAG1) but no hypermutation
• oft invoked as possible explanation for neuronal
  diversity by analogy to antibody diversity
• e.g. how can genome encode receptors for 1000s of
  odorants?
• recombinase RAG1 is expressed in the nervous
  system.

18
descriptive evidence for genomic constancy

• Beermann (1950s)
• polytene chromosomes in insects (chironomid
  midges)
• banding pattern is the same between tissues
• giant puffs (Balbiani rings) vary with time and
  tissue
• sites of active transcription (can induce with
  ecdysone)
• conclusion same DNA, differences in expression

19
functional evidence for genomic constancy
Fig 7.4

• show that a single differentiated cell can be
  induced to make entire organism (totipotency)
• Fred Steward, 1964 grow callus from single cell
  (protoplast, after removal of cell wall), then
  grow into plant.
• Cell clusters undergo morphogenesis to resemble
  early plant embryos

20
Plants are different

• long known that parts of plants can generate
  entire plant (cuttings, grafts)
• clone (klwn) greek for twig
• Plants are fundamentally different in that most
  cells in most species retain totipotency
• soma-germline distinction is fuzzy
• does not answer question for animals

21
cloning

• clone set of genetically identical organisms
• gene cloning isolate DNA that encodes a gene,
  replicate DNA in bacteria
• clone the set of identical bacteria (or the
  set of identical DNA molecules)
• clonal analysis set of genetically identical
  cells in an animal derived from mitotic
  recombination
• animal cloning

22
are animal cells totipotent?

• early blastomeres are totipotent
• e.g. Spemann (1902), split salamander 2-cell
  stage
• both cells give rise to complete animal
• loss of totipotency during embryogenesis WHY?
• do cells lose genetic material?
• or do they just become unable to express it?
  (cytoplasm becomes inhibitory)

23
early tests of totipotency

• Robert Briggs and Thomas King (1952)
• leopard frog, Rana pipiens
• developed nuclear transfer (NT) technique

24
nuclear transfer (NT)
maternal haploid pronucleus
donor cell (diploid)

• Get unfertilized oocyte
• Activate oocyte by pricking--mimics fertilization
• Remove or inactivate (by UV) maternal pronucleus
• Inject nucleus from donor cell into enucleated
  egg (no donor cytoplasm)

donor nucleus in host egg
25
results from blastula nuclei

• 60 of nuclear transfers yielded viable frogs
• genetically identical clones
• nuclei from post-tadpole stage dont work

Fig 9.30
26
serial nuclear transfer

• John Gurdon (1960s--present), working on Xenopus
• try to adjust nuclei in steps
• use nuclei from tadpole gut
• first transfers undergo partial cleavage then
  stop many aneuploid nuclei
• take best-looking nuclei and do second transfer
• result 7 of secondary transfer embryos develop
  to adult
• conclusion gut cells are totipotent

technical improvement use genetic markers to
distinguish donor derived nuclei from host
27
caveats

• Are nuclei really from gut? (could they be from
  nearby germline, so not differentiated?)
• Laskey Gurdon 1972 cultured epithelial cells

occasionally
Fig 9.29
28
Conclusions from amphibian cloning experiments

• Differentiated cell nuclei, if not totipotent,
  are highly pluripotent
• can make a lot, but maybe not everything
• therefore differentiated cells probably have same
  DNA as germline
• problem is that nucleus cant adjust fast
  enough to cytoplasmic environment of rapidly
  dividing blastula

29
Cloning mammals

• eggs are 1/10 the size of frog eggs
• development requires reimplantation into mother
• early blastomeres shown to be totipotent, but
  later loss of totipotency
• Breakthrough in 1997 cloning of Dolly (Ian
  Wilmut)

30
Dolly

• first mammal cloned by NT
• donor nuclei from mammary epithelium, in G0
  phase--quiescent
• genetic markers distinguished donor vs host
• 1 in 277 an anecdote, not an experiment?

31
Cloning state of the art 2005

• Mammalian cloning now done in many species
• clones from fully differentiated cell types
• reprogramming of epigenetic marks is the
  rate-limiting step

32
Why is the success rate so low?

• In mice
• nuclei from somatic cells 0.5 of transfers
  develop to adult
• nuclei from oocyte cumulus cells 1-2
• nuclei from ES cells 15
• does low success rate reflect problems in nuclear
  reprogramming?
• or are differentiated cells really not
  totipotent, and rare viable clones due to
  contamination

33
evidence that differentiated cells totipotent (1)

• Hochedlinger Jaenisch 2002
• get nuclei from mature B cells in which antibody
  genes rearranged
• result cloned mice in which all cells have same
  rearranged antibody genes (monoclonal)
• a differentiated cell can be totipotent
• caveat--mature B cells are not post mitotic..

34
evidence that differentiated cells totipotent (2)

• Eggan et al 2004
• Olfactory (smell) neurons as nuclear donors
  definitely post mitotic
• Cloned mice are normal. (Kills the idea that
  olfactory receptor diversity has anything to do
  with DNA rearrangement)

Sleeper, 1973
35
epigenetic reprogramming

• Major challenges to getting nuclei to readjust to
  new cytoplasmic environment
• heritable changes in chromatin structure
• heritable DNA modifications (methylation)
• programming occurs during development/differentiat
  ion and must be removed to get back to embryonic
  state

36
Reprogramming of Oct4 gene

• Byrne et al 2003
• transfer mouse thymocyte nuclei into Xenopus
  oocytes, Oct4 gene activated after 4 days
• If Oct4 plasmid DNA injected, activation
  immediate
• is delay due to DNA modification or protein
  (chromatin)?
• if deproteinated thymocyte DNA injected,
  activated in 2 days
• conclusion?

37
epigenetic reprogramming at Oct4

• Simonsson Gurdon 2004
• analyze methylation patterns at Oct4
• show demethylation after nuclear transfer, by
  as-yet unidentified DNA demethylase enzymes
• demethylation may be required to open up
  chromatin

38
methylation in early development

• both maternal and paternal haploid DNAs are
  methylated
• early embryo has active demethylase
• only paternal DNA sensitive, gets demethylated
• in nuclear transfer from somatic cells, both
  genomes sensitive, so lose parental
  imprintscould be a big problem

39
Summary nuclear programming

• Differentiated somatic cells have fixed gene
  expression patterns due to chromatin, methylation
  etc
• germline also has specific programming, but early
  embryo removes a lot of the marks
• cloning success depends on ability to reprogram
  nuclei

40
cloning via ES cells

• Recent successes in cloning from differentiated
  cells used a two-step procedure
• NT from somatic cell into enucleated egg
• allow to develop to blastocyst
• then dissociate and culture cells in vitro to
  select for proliferating ES cells
• now inject these ES cells into host blastocyst,
  classic injection chimera technique

41
Making ES cells

• all ultimately from ICM of blastocyst (Oct4)
• Oct4 expression declines after culture in vitro
• small start dividing, turn on Oct4, now
  immortal and make an ES cell line
• seem to lose epigenetic marks of early embryo or
  donor nucleus

42
problems with NT clones

• gt98 die for various random reasons
• 2 that survive often have immune or other
  dysfunctions frequently obese
• subtle/long term problems in reprogramming/lack
  of imprinting

43
Are clones born old?

• Somatic cells senesce
• accumulation of mutations, despite repair
• shortening of telomeres
• Dollys telomeres started short and she died
  prematurely (2003, age 6)
• but other clones (cows, mice) show re-setting
  of telomeres in early embryo

44
Other issues

• could mismatched nuclear and mitochondrial
  genomes cause problems? we dont know
• heterospecific transfers (Loi et al)
• mouflon (wild sheep) are endangered
• inject mouflon nuclei into domestic sheep egg
• same genus, different species
• a way to save endangered species?

The mouflon, Ovis orientalis musimon
45
there are natural animal clones

• Parthenogenesis found in
• social insects (drone bees)
• whiptail lizards
• Bdelloid rotifers (60 million years without sex)
• monozygotic twins (share nuclei, mitochondria,
  cytoplasm--unlike NT clones)

Cnemidophorus uniparens the desert grassland
whiptail